Cutaneous melanoma represents a significant and growing public health burden in the USA. This extremely deadly disease arises as a result of the acquisition of a series of genetic mutations in the melanocytes located in the bottom layer of the skin's epidermis. Although solar UV radiation is the primary cause of cutaneous melanoma, how UV causes the melanomagenesis mutations is poorly understood. Indeed, UV signature mutations, which are primarily caused by cyclobutane pyrimidine dimers (CPDs) directly induced by UV, are common in some mutated genes implicated in cutaneous melanoma. However, the UV signature mutations account for less than 10% of all melanomagenesis mutations. Therefore, DNA lesions indirectly induced by UV, such as the varieties of oxidative lesions and N-methylpurines (NMPs), and/or those induced by other as-yet-unidentified DNA damaging agents may be responsible for the majority of the melanomagenesis mutations. Our long term goal is to gain a deeper understanding of the etiological causes of melanomagenesis mutations and how DNA damage and repair are implicated in the mutagenesis process. Systematic nucleotide-level mapping of induction and repair of diverse types of DNA lesions in human melanocytes can offer invaluable insights into the causes of the melanomagenesis mutations. The major roadblock to this task is that all currently available methods for DNA damage and repair mapping lack the resolution, sensitivity and/or throughput. This proposal contains two specific aims. In aim 1, we will develop a novel extremely sensitive method for high-throughput nucleotide-level mapping of DNA damage and repair in living human cells. The next- generation DNA sequencing technologies will be utilized for the development of the novel method. In aim 2, we will map DNA damage induction and repair related to melanomagenesis mutations in human melanocytes. The induction and repair of various types of DNA lesions, including CPDs, oxidative lesions and NMPs, will be mapped in melanocytes from people of different skin types. The genomic regions we plan to map include the sites of melanomagenesis mutations and their neighboring sequences. The correlations of the DNA damage induction and repair events with the site-specific melanomagenesis mutations will be systematically assessed. The tendencies of the identified lesions to form the site-specific mutations will be further confirmed by using a technique that can detect a single mutation among millions or billions of wild type DNA molecules. PUBLIC HEALTH RELEVANCE: This project addresses the long standing question regarding how DNA damage and repair are implicated in the generation of melanomagenesis mutations. Accomplishment of the proposed studies will offer invaluable insight into the etiological causes of melanoma and be very informative for development of strategies for preventing the devastating disease.